You need to get calories from somewhere, should it be from carbohydrate or fat?

Friday, November 04, 2011

Metabolic flexibility and the identical twins

This post is highly speculative. It doesn't have any answers. Here is a nice quote to begin with:

"If you want to retain your sanity, don't try to read a textbook on mitochondrial diseases"

This is from Nick Lane on page 281 of Power, Sex, Suicide. I was going to copy out the preceding paragraph but I guess everyone has their own copy of PSS. If not, you know what to do.

Now think about your sanity if you are dealing with a problem like obesity and you don't accept it's mitochondrial... Also think about the likelihood of successful intervention.

So I'm putting this up as a one-liner-which-grew because Liz dropped this paper me a few days ago and I got chance to open it today (OK, over a week ago!).

Enrol monozygotic twins in Finland. Hunt out BMI discordant identical twins (they are very rare) from the study, ie pairs of genetically identical people where one gets fat and one doesn't, despite their identical nuclear genes. Do lots of studies, get a Nature publication or ten out of it and decide obesity occurs because folks eat too much and move too little. Go to the top of the class as obesity researchers. There's a lot of it about.

Let's pick through the discussion and look at some of the conclusions from the metabolic flexibility point of view:

"a slightly higher birth weight (193 g) was observed for the twin that developed obesity during early adulthood, but this difference disappeared by age 6 months and the growth patterns of both twins were virtually identical until the age of 18 years, after which BMI differences between the co-twins became statistically significant (Figure 3)."

Pre-obese half of the pair of twins were heavier at birth, ie heavier in-utero. They must have been sneaking out to Macdonalds while telling their mother they were off to the gym. Amazing what some pregnant women will let their foetuses get up to. Next:

"After age 8, the pairs who later became discordant for obesity were heavier than the population mean, raising the possibility that genetic or environmental factors predisposing to obesity may be present in both co-twins of the discordant pairs. It therefore remains an open question as to whether the lean or the obese co-twin actually is more closely following the genetic predisposition."

Both twins have identical nuclear genes. These may or may not predispose to obesity, who knows? The obese twin has more defective mitochondrial genes than the one who remains slim. Each followed their need to produce adequate ATP. The one with worst mitochondria had to become obese to get there. Even the "slim" twin was heavier than average. His mitochondria might not have been so hot either, but not bad enough for serious malfunction. Next:

"The results suggested that physical inactivity in adolescence strongly predicted the risk for obesity (OR 3.9) and abdominal obesity (OR 4.8) at age 25, even after adjusting for baseline and current BMI."

Physical activity in adolescence is difficult if you have inadequate ATP production, so is minimised. At this age the affected twin is pre-obese. Obesity is necessary for elevation of FFAs to a level which will normalise ATP production to allow normal physical activity with sub normal mitochondria. Insulin will raise fat depots to an adequate size to elevate FFA supply due to adipocyte insulin resistance, once childhood growth has ended. Next:

"At age 25, the obese co-twins were only half as active compared with their lean co-twin as demonstrated in the 7-day accelerometer measurements.31 However, the total energy expenditure and activity-induced energy expenditure from the doubly labelled water did not differ between the co-twins. This discrepancy may be explained by the fact that the obese twins, while moving on average less, do expend more energy when they do because of their higher body weight."

THERE IS NO DIFFERENCE IN ACTIVITY OR CALORIE INTAKE BETWEEN TWINS ONCE OBESITY IS ESTABLISHED. An obese person moving from standing to sitting to standing again is doing a much weightier squat than the equally-idle-but-apparently-active skinny person with no fat to lift. Fatties may look idle because they don't get up from their chair if they don't have to but THERE IS NO DIFFERENCE in energy expenditure AT ALL compared to those equally "lazy" skinny twins who get up a few more times to burn the EXACTLY the same calories. OK, I've stopped shouting now. Doubly labelled water. Next:

"The basal metabolic rates (as measured by calorimetry) were considerably higher in the obese co-twins, presumably for the same reason."

Repeat shouting from previous paragraph. Plus, oops, they could have been talking about the Pima and forgot to mention that post prandial thermogenesis was depressed by almost exactly as much as BMR was increased.... Heard that before? I've not gone in to the logic of what is happening to BMR vs post prandial thermogenesis but it will undoubtedly come down to mitochondrial function. It just amused me that these established stars of obesity research were so familiar in their technique of citation. Next:

"The prospective Norfolk study of 20 000 men and women showed physical activity to attenuate the genetic predisposition to common obesity by 40%, as estimated by the number of risk alleles carried for 12 recently identified obesity predisposing loci.34 In the same study, the genetic risk score was positively associated with weight gain in inactive subjects, but negatively associated in physically active subjects."

No no no no. This appears to be saying that certain nuclear genes are associated with obesity if you are lazy. HOWEVER exactly the same genes are associated with you being THIN if you are active. I've not chased the EPIC paper because it's pure observational stuff but that's what this quote appears to claim EPIC is saying. Correct me if I am wrong. One explanation is that they are looking at the wrong set of genes. Obesity is a mitochondrial disease. It doesn't matter too much what your nuclear DNA says. You need good mitochondria to allow you to be physically active without needing you to be obese to improve ATP production. Duff mitochondria only allow you to be active if you have accumulated enough adipose tissue to trickle out FFAs. Next:

"However, the more objective measures via doubly labelled water revealed a substantial reporting bias by the obese co-twins: the under-reporting of energy intake (3.2±1.1 MJ per day) and over-reporting of physical activity (1.8±0.8 MJ per day) in the obese twins equalled to as much as one Big Mac hamburger, a 16-oz bottle of soft drink and almost 90 min of walking (3 m.p.h.), respectively. Interestingly, however, when asked to compare their own eating habits and physical activity to those of their co-twin, both co-twins openly reported that the obese co-twin had an unhealthier lifestyle with overeating, snacking and an irregular eating pattern as well as less physical exercise (Figure 4)."

This is a lovely paragraph. I think I have to accept from doubly labelled water that fatties lie about their caloric intake. This is very surprising. By doubly labelled water fattie twins do NOT eat any more than slim twins. They do not exercise less. Calories in and calories out are IDENTICAL in the obese and slim halves of the pair. Why should the fatties lie and claim to eat less than their skinny twin? Because they're fat...

I think it is also worth saying that the obesity-destined twin was noted, by all and sundry, to be "overeating, eating badly and eating irregularly" from an early age, with a preference for fatty foods. However I would comment that they did not even begin to become obese until 18 years of age and by 25 years of age doubly labelled water showed... etc etc etc. This moral failing as youngsters might just be translated as the pre-obese half of the pair were HUNGRY at that time. Life is hard when the world views your moral failings at the snack bar as evidence of your lack of will power. Being hungry is no fun. Being hungry because your adipocytes are not fat enough (yet) to ignore your hyperinsulinaemia and let you, grudgingly, have a few FFA molecules from their hoard is somewhat unfair. Your skinny twin is not hungry. He has mitochondrial ATP to spare. He sniggers at your third helping of pizza at your 18th birthday party because he has no gnawing hunger. He knows that you lie about how much you eat by your 25th birthday party. But by then he is eating EXACTLY the same as you are... At the gym, where he is well known, he only burns as many calories as you do walking up stairs. DOUBLY LABELLED water. Life is unfair. Next:

"Environmental influences independent from acquired obesity on liver fat were evaluated based on questionnaires and food diaries. Alcohol consumption from detailed questionnaires of the obese (3.7±0.9 doses per week) and non-obese (3.9±1.1 doses per week) co-twins did not differ and intra-pair differences in alcohol intake did not significantly correlate with differences in liver fat (r¼0.30, P¼0.14). Analysis of data from food diaries showed that the percentage of energy from fat (r¼0.37, P¼0.02) and saturated fat (r¼0.38, P¼0.005) did correlate with liver fat.5"

It gets better. The same group looked at fat preference. They really looked at fat preference. Not Fanta preference. They ONLY looked at fat preference. Perhaps there was no Fanta preference, it's not needed if the damage is already done. But the abstract gives no suggestion that they looked at anything other than fat... What answer did they set out to find? As I mentioned, there's a lot of it about.

Here's the scenario. Both twins get home from school. Pre-obese is hungry. Sneaks in to pantry and finds... Dadahhhh, a block of butter! You believe he skipped on the cookies sitting there?

Monozygotic twins have identical nuclear genes. They normally have very similar mitochondrial genes. But if there is mitochondrial heteroplasmy in the oocyst and one twin gets a bigger share of the decent mitochondria while the other gets a duff lot as they separate in-utero, things will be different. There will a discordance in BMI which develops in the attempt to normalise ATP production in the obese twin. The pre-obese twin is pre-obese in utero.

This would all be hunky dory if the mitochondrial heteroplasmy existed, with differing mitochondrial mutations between the twins. It doesn't, apparently. We find this snippet towards the end of the review paper:

"A novel finding of great interest in our obesity-discordant MZ pairs was the dramatic reduction of copies of mitochondrial DNA in the adipose tissue of the obese co-twin.12 Although the sequence of mitochondrial sequence was identical between the MZ twins (no evidence of heteroplasmy), the copy number of mitochondrial DNA in the obese co-twin’s adipose tissue was only 53% of that of the lean co-twin."

Sorry about the odd sentence in exactly the place where we want clarity, that's just how it is. Anyway, no evidence of heteroplasmy. But let's go and look up Ref 12.

This gives us this line:

"The mtDNA sequences of fat showed no evidence for heteroplasmy in co-twins, nor potentially obesity-associated sequence changes between obese and non-obese co-twins in fat or in leukocytes (Figure S1)."

I guess this might mean (as originally cited) that the sequences were identical between obese and normal twins, but it actually says there were no "potentially obesity-associated sequence changes between obese and non-obese co-twins", which may or may not be the same thing.

The next move is to another supplementary document which gives us this text (you don't have to read it if you don't want to):

"Analyses of mitochondrial sequence and copy-number

Known mitochondrial DNA sequence variants were extracted from MITOMAP database (www.mitomap.org) and variant information was annotated to the selected reference sequence AC000021.1 (GI:58615662) from GenBank. PCR primers were selected and re-optimized among those presented by Sigurdsson et al 7. Sequencing primers were designed to avoid known variant positions using The PCR Suite 8. The mitochondrial genome was PCR amplified in two overlapping ~9 kb fragments. PCR amplification was performed using 20-30 ng of DNA, 14 pmol each primer, 200 μM dNTP 1,4 U of DyNAzyme EXT DNA polymerase in 1X DyNAzyme EXT buffer (Finnzymes). Thermocycling consisted of denaturation of DNA template in 94ºC for 2 min followed by 30 cycles of 94ºC for 20s, 60ºC for 30s and of 72ºC for 4 min (extended for 10 s / cycle) and final extension of 72º for 15 min. Correct amplification was verified by agarose gel electrophoresis. PCR products were ExoI / SAP purified and sequencing was performed with BigDye3.1 chemistry on an ABI 3730xl DNA Analyzer. Mitochondrial consensus sequences and sequence variants were determined with SeqScape Software v2.5 (Applied Biosystems). Oligonucleotide sequences used in PCR and sequencing are presented in the Appendix of Supplementary Methods (vide infra)."

This is, to my rather limited experience, a standard PCR and sequencing protocol and is essentially guaranteed to produce mtDNA homoplasmy. Why? The number of abnormal mtDNA sequences is low amongst a huge number of normal copies. If you want to find heteroplasmy you have to individually sequence lots and lots and lots of mtDNA strands. Running a standard sequencing machine will not hack it.

"Here, we describe digital sequencing of mtDNA genomes using massively parallel sequencing-by-synthesis. Though the mtDNA of human cells is considered to be homogeneous, we found widespread heterogeneity (heteroplasmy) in the mtDNA of normal human cells. Moreover, the frequency of heteroplasmic variants among different tissues of the same individual varied considerably"

I've struggled with the methods to this paper and I'm not sure how many mtDNA strands they sampled from a given tissue. I think they might have done quite a few. This paper adopted a similar approach. Looking this hard you tend to find heteroplasmy if it is there.

It's there.

There are some interesting ideas in both papers about how heteroplasmy gets in to various tissues at various levels but they didn't go so far as to consider identical twins with mismatched phenotypes. A pity, because I think they know a great deal more about this than I do.

An obese twin has only 53% of the mtDNA of the slim one in their adipocytes. How about muscle cells? We know from the EMs of insulin resistant offspring of diabetic parents that their muscle mitochondria are grossly abnormal. We find from the twins study that lots of mtDNA (and presumably the mitochondria which might have originally contained it) simply isn't there.

It must be rather hard to find the mtDNA of mitochondria which are not there. Or mtDNA which is only there in very small copy numbers in the surviving mitochondria.

I personally doubt the mtDNA was homoplasmic in the obese twins. The unanswerable question is whether the heteroplasmy is responsible for the decreased mtDNA count...

There are a whole stack of ideas from the twins papers which need looking at from the mitochondrial selection pressure perspective, what controls mitochondrial number and how mitochondria control nuclear genes for their own synthesis...

Peter

BTW, compare these two abstracts, both from Finland Twins studies group:

20 comments:

Might it not be that the obese twin was doomed due to the drawing of a bad lot of mtDNA and actually arrived there through malnutrition in the first 18 years? Couldn't the obese twin have a chromium/magnesium/manganese/zinc/biotin/b5/carnitine/carnosine/ubiquinol/PQQ/ALA (all necessary for proper mitochondria function/biogenesis) deficiency that he developed over nearly two decades of choosing poorer food choices than the other twin?

The do seem to acknowledge the primary role of mitochondrial dysfunction without explicitely postulating the heteroplasmy. The following quote from that seems to be implying it (unless I am misinterpreting):

Lending clinical relevance to the findings, in both sexes the observed aberrations in mitochondrial amino acid metabolism pathways in fat correlated closely with liver fat accumulation, insulin resistance, and hyperinsulinemia, early aberrations of acquired obesity in these healthy young adults.

CONCLUSIONS:

Our findings emphasize a substantial role of mitochondrial energy- and amino acid metabolism in obesity and development of insulin resistance.

What else other than heteroplasmy can produce differences in mitochondrial activity in MZ twins at all ages? Could some acquired condition such as a viral infection, toxins or accidental overfeeding domage one twin's mitochondria but not the other one?

so i took a loooong look at Power, Sex, Suicide. . Some chapters are engaging, others... wtf ( my English @patience is not the best btw)

particularly enjoyed the part about aging, and caloric restriction as the only proven mechanism to extend the lifespan in animals...( free radical restriction and all) so instead of fumbling with dat macros. better start fasting guys ...

lol you can taste the desperation of the author, bu bu the rate of engerited mitochondrial mutations i want to be a yeast! i dont want to die!! ... lol thanks peter great read

Stan and Travis, I was really kicking the heteroplasmy idea around to see if it might fit the pattern. As with all of the information on obesity you can look at it from the end of hypernutrition being the problem and you don't need a primary mitochondrial problem. In which case you end up with some derivative of the reward hypothesis. Indeed, an absence of heteroplasmy drives you in this direction, as the Findland twins studies authors have concluded, though they clearly view mitochondrial issues as important secondarily.

Interestingly both twins are underweight cf population means, as you would expect due to sharing their mother's nutrient supply. As slightly low birthweight children, both end up slightly above the population mean, again as is normal worldwide. But of the two twins the one which is heaviest at birth (by about 200g out of a lower-than-average birthweight), the heaviest twin was the one to become obese as an adult, the opposite of the normal effect of birthweight on adult weight.

I had wondered, as my wife pointed out, about differences in placentation being a significant factor (some monozygotic twins have significant plancental oddities) but if the effect is epigenetic due to reduced nutrition I would have expected the smaller twin to become the obese one.

Of course there may be no mitochondrial heteroplasmy, but so far no one has looked in any depth...

Again Peter, very out-of-the-box thinking, nice. But you think faster than you type! it takes me a while to fully comprehend the gravitas of such statements as: “Each followed their need to produce adequate ATP. The one with worst mitochondria had to become obese to get there.”[… repeat with ~6 more examples before the end of the post …]

Seems like a fairly easy explanation might also simply be that one of the twins, for whatever reason, decided somewhere along the way to be less active. Mitochondrial density in the muscles would rapidly atrophy and glucose tolerance would suffer significantly. Reading about mice so much we forget that humans can decide to be lazy at any moment and their health will go to Hell as a result.

Peter, You said:You need good mitochondria to allow you to be physically active without needing you to be obese to improve ATP production. Duff mitochondria only allow you to be active if you have accumulated enough adipose tissue to trickle out FFAs.

Would you mind explaining this a little further to a simple "Taubsian"?

I think I get the bit about needing adipose tissue tricking out FFAs (because of insulin resistance in the adipocytes, so they can't store any more fat, I believe).

What I've never quite got about this aspect is that if we are insulin resistant in our adipocytes, and gushing out FFAs ("lipotoxicity" as I believe the jargon has it), should these FFAs not be then available as an energy source (and so we shouldn't feel hungry, "overeat", and get even fatter?

According to Gary, it's because high insulin is keeping fat locked into our adipocytes, and not letting out FFAs, that's depriving our cells of energy, making us fatties feel hungry all the time, and wanting to eat everything that's not tied down.

So why would it be such a bad thing then, if our adipocytes go insulin resistant and can't store any more fat, and FFAs float around in circulation?

Is there a difference between FFAs floating around under these circumstances, and FFAs which become available when we have normal insulin levels and our fat is not "locked in" to our adicpocytes? Sorry, probably a naive question, but it's been bothering me.

And where then, please, does good and bad mitochondria, and ATP fit in?

P.S. Actually, I have just realised you may have answered this already, in your earlier article about the adipostat(sp?) balloon. I did read that (or at least skim read it) before, but maybe my brain cells were low on ketones at the time or something.

Brash indeed, however you may have missed this: "the growth patterns of both twins were virtually identical until the age of 18 years, after which BMI differences between the co-twins became statistically significant."

Stan said "What else other than heteroplasmy can produce differences in mitochondrial activity in MZ twins at all ages? Could some acquired condition such as a viral infection, toxins or accidental overfeeding domage one twin's mitochondria but not the other one?" This also ties in with Travis' and Ted's comments;imagine for whatever reason one twin is given antibiotics early on and the other is not. Alterations in gut microbiota result in micronutrient deprivation, sensitisation to gluten, or any number of changes influencing weight. Antibiotics are used to fatten cattle.Exposure to antibiotics is one of the variables I'd like to see measured; also, differences in communal living, as in boarding schools or hospitals, which would also affect microbiota.Vaccination schedules too. Immune activation against pathogenic bacteria will likely impact on some non-target species.

About Me

I am Petro Dobromylskyj, always known as Peter. I'm a vet, trained at the RVC, London University. I was fortunate enough to intercalate a BSc degree in physiology in to my veterinary degree. I was even more fortunate to study under Patrick Wall at UCH, who set me on course to become a veterinary anaesthetist, mostly working on acute pain control. That led to the Certificate then Diploma in Veterinary Anaesthesia and enough publications to allow me to enter the European College of Veterinary Anaesthesia and Analgesia as a de facto founding member. Anaesthesia teaches you a lot. Basic science is combined with the occasional need to act rapidly. Wrong decisions can reward you with catastrophe in seconds. Thinking is mandatory.
I stumbled on to nutrition completely by accident. Once you have been taught to think, it's hard to stop. I think about lots of things. These are some of them.

Organisation (or lack of it)!

The "labels" function on this blog has been used to function as an index and I've tended to group similar subjects together by using labels starting with identical text. If they're numbered within a similar label, start with (1). The archive is predominantly to show the posts I've put up in the last month, if people want to keep track of recent goings on. I might change it to the previous week if I ever get to time to put up enough posts in a week to justify it. That seems to be the best I can do within the limits of this blogging software!